The majority of mature excitatory synapses in the CNS occur on specialized morphologic structures known as dendritic spines, and it is clear from studies in cell culture that the EphB receptor tyrosine kinase and the NMDAR are involved in proper spine and synapse formation. Recent work has also shown that activated EphB can both recruit and modulate the function of NMDARs. Aim 1 of this proposal will determine the steps of dendritic spine morphogenesis that are regulated by EphB receptor signaling by examining and manipulating neurons over time from EphB null mice. Aim 2 will assess the ability of activated EphB to recruit NMDARs in a more complex system, cortical slice culture, using fluorescently-tagged receptors and two-photon microscopy. Finally, the known interaction of EphB and NMDARs suggests a coordinated role during the development of dendritic spines; Aim 3 will directly test for this cooperative activity in cortical slice using pharmacology to manipulate receptor function and two-photon imaging to visualize spine morphology. Increased understanding of these molecular mechanisms is critical in addressing human neurologic diseases whose phenotype include abnormal spine formation, such as Fragile X, Down, and Rett Syndromes.